Background: Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin–thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the “Paris” mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC).

Material and Methods: We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation.

Results: We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (²⁵²NTT²⁵⁴) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC.

Conclusion: The new N-glycosylation site (²⁵²NTT²⁵⁴) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

背景： 蛋白 C (PC) 是一种维生素 K 依赖性抗凝丝氨酸蛋白酶酶原，在被凝血酶-血栓调节蛋白 (TM) 复合物激活后，通过有限的蛋白水解降解辅助因子 Va 和 VIIIa，从而下调凝血级联。我们在PROC中鉴定出一名血栓患者携带 c.881G > A、p.Ser252Asn (S252N) 杂合突变。这种突变最初是在巴黎一份 PC 抗凝活性缺陷患者的新突变报告中描述的。该研究在一名产妇及其家庭成员中发现了 PC-S252N（“巴黎”突变），并强调了 Ser252 在活化 PC (APC) 抗凝过程中的关键作用。

材料和方法： 我们在哺乳动物细胞中表达了 PC-S252N 突变体，并表征了凝血测定中的特性，以破译该突变的抗凝血缺陷的分子基础。

结果： 我们证明 PC-S252N 与 TM 结合的能力减弱，导致其被凝血酶-TM 复合物激活受损。然而，APC-S252N 对显色底物表现出稍强的裂解能力。同时，APC-S252N对FVa的催化活性显着降低。序列分析显示，Ser252 替换为 Asn，在 PC 的催化结构域中引入了一个新的潜在 N 连接糖基化位点 ( ²⁵² NTT ²⁵⁴ )，这对 PC 的激活过程和 APC 的抗凝活性产生不利影响。

结论：PROC中Ser252突变为Asn252产生的 新的N-糖基化位点（²⁵² NTT ²⁵⁴ ）影响了蛋白酶的整体结构，从而对Protein C的抗凝功能产生不利影响。这种修饰对两种TM均产生负面影响-促进蛋白C的激活和APC对FVa的裂解，最终导致患者血栓形成。